In the coming year we will continue development of methods to study axonal transport in the central nervous system of the rat. Procedures utilizing sodium diatrizoate gradients will be further modified to obtain purified nerve ending membrane and nerve ending vesicle fractions. Kinetics of transport of glycoproteins (labeled with radioactive fucose or glucosamine) and of sulfated glycoproteins (35S-sulfate labeled) will be obtained and the proteins characterized by two dimensional gel electrophoresis techniques. Double isotope techniques (time staggered injection of first a (3H)labeled precursor and then a (14C) labeled precursor in the same chemical form) will be developed to study the relative time sequence of incorporation of macromolecules into these nerve ending subfractions. The kinetics of this process will be studied in rats during development (between 10 days and 60 days of age). This methodology, and that developed previously with respect to isotope methods for studying the sequence of incorporation of proteins into the myelin sheath (Benjamins, et al., 1976, J. Neurochem. 27, p. 571), will be used to search for perturbations in metabolism of brain membranes in rats undergoing various metabolic insults. Among the models studied will be rats made hypothyroid by injection of methimazole and rats made hyperthyroid by injection with thyroxine. BIBLIOGRAPHIC REFERENCES: Truding, R. and Morell, P. (1977) Effect of N6, O2' Dibutyryl Adenosine Cyclic 3':5'-monophosphate on the Release of Surface Proteins by Murine Neuroblastoma Cells, J. Biol. Chem., 252, in press. Miller, S.L., Benjamins, J.A., and Morell, P. (1977) Metabolism of Glycerophospholipids of Myelin and Microsomes in Rat Brain. Reutilization of Precursors. J. Biol. Chem., in press.